bacteria identification & classification

Question 1

what are the 3 steps for identifying bacteria for the bacteriology laboratory?

Answer 1

1. correct sampling procedures & provision of adequate history
2. transport to lab to ensure viability of suspect organism & minimal contamination
3. growth, identification & antibiotic sensitivity testing

Question 2

what does the correct sampling procedure step for identifying bacteria involve?

Answer 2

• obtaining the specimen aseptically from the disease site
• collecting the specimen at the correct time during disease process (acute stage of infection)
• if collecting sample from a dead animal - collect sample before post-mortem changes occur & from site most likely to yield pathogens

Question 3

what information is collected for the provision of an adequate history?

Answer 3

collection of a full clinical history

• age, sex, species, number of animals affected (if production animal)
• treatment administered
• tentative clinical diagnosis

Question 4

what does the transport to the lab step involve?

Answer 4

• involves ensuring viability of suspect organism & minimising contamination
• transport media should be a semi-solid agar containing salts & buffers
• keep sample at 4*C
• body fluids other than urine should be held at room temperature
• submitting sample in leak proof containers
• simple should be labelled & delivered to lab within 48hrs

Question 5

once sample is transported to the lab, how do you choose what media to grow bacteria on?

Answer 5

if suspecting:

• mucosal infection - use blood agar & MacConkey agar
• anaerobic bacteria from body fluid sample - add a second agar plate incubated anaerobically
• fastidious organisms - use chocolates or specialised agar
• fungal pathogens - add Sabouraud’s agar plate plus chloramphenicol

Question 6

what does the growth, identification & antibiotic sensitivity testing step involve for identifying bacteria?

Answer 6

1. choose media, plate out & incubate
2. initial smear on gram stain is the first step in identification of a bacterial organism - guides choice of initial therapy
3. examine plates every 24 hours to look at colony morphology on solid media
4. obtain a pure culture - as bacterial identification can only be performed on pure cultures of bacteria (all descendants from one bacterial cell)
5. perform a minimum number of tests to identify the isolate to a species level
   - tests mostly biochemical, some microscopic, immunological or molecular
6. determine antimicrobial sensitivity profile

Question 7

how is a pure culture obtained?

Answer 7

• spread bacteria on an agar plate to achieve individual bacterial colonies from a single cell
• a large amount of organism in PURE culture is required to do more tests

Question 8

when is solid media chosen and when is broth chosen as the media to grow organisms on?

Answer 8

• generally both liquid & solid media are innoculated for microbe isolation
• solid media - grows individual colonies, allowing quantification of bacteria & differentiation of normal flora from potential pathogen
• broth - allows recovery of a small number of organisms or those that are more fastidious

Question 9

define a bacterial strain

Answer 9

• a line of bacteria descended from a single ancestor through various subcultures

Question 10

define an isolate?

Answer 10

• is each primary culture isolated from a natural source

Question 11

what do biochemical tests detect when we use them to identify bacteria?

Answer 11

• detect enzyme activity
• detect end products to identify substrate utilisation
• eg rapid kits, oxidase test, catalase test

Question 12

what do immunological tests detect when we use them to identify bacteria?

Answer 12

• detect antigen-antibody reactions (agglutination reaction)
• clumping indicates presence of antigen related to a specific species of bacteria
• used to identify bacteria sterotypes/serovars

Question 13

define serotypes

Answer 13

• are groups of microorganisms grouped together based in their cell surface antigens

Question 14

why would a molecular test be chosen as the method to identify bacteria?

Answer 14

can be used to identify bacteria that:

• cannot be cultured
• are slow or difficult to grow
• when rapid diagnosis is important
• can be used to measure antimicrobial resistance

Question 15

what are the 3 approaches of molecular tests that can be used to identify bacteria?

Answer 15

1. immunological - includes
   - serology of surface antigens
   - or detecting other markers
2. chemical/physical
   - includes simple indirect tests
   - or MALDI-TOF
3. genetic
   - PCR
   - genome sequencing
   - molecular typing

Question 16

what can PCR be used for?

Answer 16

• identifying bacteria to a species level
• identifying strain
• characterising bacteria (eg virulence genes)
• real time PCR gives instant results & can estimate initial bacterial load

Question 17

what is MALDI-TOF used for?

Matrix Associated Laser Depolarisation/Ionisation-Time of Flight

Answer 17

• MALDI-TOF is a form of matrix spectrometry that identifies bacterial species
• high capital cost but low operation cost
• only as good as reference database

Question 18

what is molecular typing used for?

finger-printing of bacteria using genomics

Answer 18

• determining matching strains for outbreak investigations
• determining phylogenetic relationships
• identifying sub-species & sub-types

Question 19

what is whole genome sequencing detect during bacteria identification?

Answer 19

• can detect single nucleotide polymorphisms
• allows better comparisons between labs
• provides evolutionary info

Question 20

what are the limitations to molecular methods that can be used to identify bacteria?

Answer 20

• cost & availability
• design & validation of the procedure
• quality of data base library
• accuracy & viability of interpretation
• some level of culture is often still required to get enough info
• no isolate in sample

Question 21

how are bacteria classified?

Answer 21

• bacteria classification is based on a set of morphological, biochemical & genetic characteristics

Question 22

what is the gold standard in bacterial taxonomy?

Answer 22

sequencing of 16S ribosomal RNA molecules

• provides genetic similarity above species level
• improves identification of some bacterial pathogens
• but increasingly being replaced by whole genome sequencing

Question 23

how are bacteria named?

Answer 23

• bacteria are given a genus name & a species name
• species levels is the most important rank in taxonomic hierarchy
• 16S RNA sequence must be >98% identical to belong in same species
• I6S RNA sequences must to be >95% identical to belong in same genus